Structural Basis of Opioid Receptor Function

说明(申请人提供)：本提案的目标是确定阿片受体药理和功能的结构基础。阿片受体是治疗疼痛的主要和最有效的靶点。然而，在欧洲和北美，作用于这些受体的阿片类药物的使用是过量死亡的主要原因。非法阿片类药物(鸦片、海洛因)以及已批准的治疗药物(吗啡和可待因)的有益和不良影响都是通过激活G蛋白偶联受体的成员u阿片受体(μ-OR)来调节的 (GPCRs)超家族。为了了解μ-OR功能的结构基础，我们获得了该受体非活性状态的第一个高分辨晶体结构，以及一个密切相关的家族成员--增量阿片受体(δ-OR)。这些结构为OR对特定拮抗剂药物的偏好提供了新的见解，但没有解决有关OR激活机制的重要问题，特别是阿片类药物的疗效。因此，我们建议使用生化和生物物理方法相结合的方法来表征阿片受体激活的结构基础。具体目标包括：目的1.确定μ-OR和δ-OR的非活性状态结构，首次揭示了吗啡类拮抗剂的结合模式。这一目的的目的是获得对G蛋白Gi阿片受体激活过程的结构洞察。我们首先将重点放在使用结晶学和单粒子电子显微镜来获得μ-OR-GI和∂-OR-GI络合物的三维结构。我们还将开发一种 一组骆驼抗体片段(纳米体)，用于稳定结晶学的配体特定构象状态。这些将被用来确定阿片受体激动剂不同功能特性的结构基础。目的2.OR结构的构象动力学与激活。或激活，更广泛地说，GPCR激活涉及配体结合和G蛋白偶联结构域之间的复杂变构偶联，但人们对此知之甚少。晶体结构提供了这一动态过程的有限数量的静态快照。因此，我们建议开发和应用生物物理方法来表征ORs的结构可塑性和动态特性，并确定这是如何转化为信号复杂性和配体有效性的。非活化态晶体结构将成为设计和解释生化和生物物理研究的重要起点，其中一些研究包括荧光、EPR和核磁共振光谱。这些研究将为阿片类配体的疗效以及小分子和多肽配体之间的差异提供新的见解。

DESCRIPTION (provided by applicant): The goal of this proposal is to determine the structural basis for opioid receptor pharmacology and function. Opioid receptors constitute the major and the most effective target for the treatment of pain. The use of opioid drugs acting at these receptors is however a leading cause of death by overdose in Europe and North America. Both beneficial and adverse effects of illicit opioid drugs (opium, heroin) as well as approved therapeutics (morphine and codeine) are mediated by the activation of mu opioid receptor (μ-OR), a member of the G protein-coupled receptors (GPCRs) superfamily. To understand the structural basis for μ-OR function, we obtained the first high-resolution crystal structure of an inactive state of this receptor along with a closely relate family member the delta-opioid receptor (δ-OR). These structures provide new insights into OR preference for specific antagonist drugs but do not address important questions regarding OR activation mechanisms and, in particular, opioid drug efficacy. We therefore propose to characterize the structural basis of opioid receptor activation using a combination of biochemical and biophysical approaches. Specific Aims include: Aim 1. Determine active state structures of ORs The inactive state structures of the μ-OR and δ-OR provided the first structural insights into the binding mode of morphinan antagonists. The goal of this aim is to obtain structural insights into the process of opioid receptor activation of the G protein Gi. We will initially focus on the use of crystallography and single particle electron microscopy to obtain three-dimensional structures of the μ-OR-Gi and ∂-OR-Gi complexes. We will also develop a panel of camelid antibody fragments (nanobodies) that stabilized ligand-specific conformational states for crystallography. These will be used to determine the structural basis for the different functional properties of opioid receptor agonists. Aim 2. Conformational dynamics of OR structure and activation. OR activation and more generally GPCR activation involves a complex allosteric coupling between ligand binding and G protein coupling domains that is poorly understood. Crystal structures offer a limited number of static snapshots of this dynamic process. We therefore propose to develop and apply biophysical approaches to characterize the structural plasticity and dynamic properties of ORs and to determine how this is translated into signaling complexity and ligand efficacy. Inactive state crystal structures will constitute an important starting point for designing and interpreting biochemical and biophysical studies, some of which include fluorescence, EPR and NMR spectroscopy. These studies will provide new insights into opioid ligand efficacy and the differences between small molecule and peptide ligands.